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# pydna Package

## `dsdna` Module

Provides two classes, Dseq and Dseqrecord, for handling double stranded DNA sequences. Dseq and Dseqrecord are subclasses of Biopythons Seq and SeqRecord classes, respectively. These classes support the notion of circular and linear DNA.

class `pydna.dsdna.``Dseq`(watson, crick=None, ovhg=None, linear=None, circular=None, alphabet=IUPACAmbiguousDNA())

  Bases: `Bio.Seq.Seq`

  Dseq is a class designed to hold information for a double stranded DNA fragment. Dseq also holds information describing the topology of the DNA fragment (linear or circular).

  Dseq is a subclass of the Biopython Seq object. It stores two strings representing the watson (sense) and crick(antisense) strands. two properties called linear and circular, and a numeric value ovhg (overhang) describing the stagger for the watson and crick strand in the 5’ end of the fragment.

  The most common usage is probably to create a Dseq object as a part of a Dseqrecord object (see Dseqrecord).

  There are three ways of creating a Dseq object directly:

  Only one argument (string):

      >>> import pydna
      >>> pydna.Dseq("aaa")
      Dseq(-3)
      aaa
      ttt

  The given string will be interpreted as the watson strand of a blunt, linear double stranded sequence object. The crick strand is created automatically from the watson strand.

  Two arguments (string, string):

      >>> import pydna
      >>> pydna.Dseq("gggaaat","ttt")
      Dseq(-7)
      gggaaat
         ttt

  If both watson and crick are given, but not ovhg an attempt will be made to find the best annealing between the strands. There are limitations to this! For long fragments it is quite slow. The length of the annealing sequences have to be at least half the length of the shortest of the strands.

  Three arguments (string, string, ovhg=int):

  The ovhg parameter controls the stagger at the five prime end:

      ovhg=-2

      XXXXX
        XXXXX

      ovhg=-1

      XXXXX
       XXXXX

      ovhg=0

      XXXXX
      XXXXX

      ovhg=1

       XXXXX
      XXXXX

      ovhg=2

        XXXXX
      XXXXX

  Example of creating Dseq objects with different amounts of stagger:

      >>> pydna.Dseq(watson="agt",crick="actta",ovhg=-2)
      Dseq(-7)
      agt
        attca
      >>> pydna.Dseq(watson="agt",crick="actta",ovhg=-1)
      Dseq(-6)
      agt
       attca
      >>> pydna.Dseq(watson="agt",crick="actta",ovhg=0)
      Dseq(-5)
      agt
      attca
      >>> pydna.Dseq(watson="agt",crick="actta",ovhg=1)
      Dseq(-5)
       agt
      attca
      >>> pydna.Dseq(watson="agt",crick="actta",ovhg=2)
      Dseq(-5)
        agt
      attca

  the ovhg parameter has to be given with both watson and crick, otherwise an exception is raised.

      >>> pydna.Dseq(watson="agt",ovhg=2)
      Traceback (most recent call last):
        File "<stdin>", line 1, in <module>
        File "/usr/local/lib/python2.7/dist-packages/pydna_/dsdna.py", line 169, in __init__
          else:
      Exception: ovhg defined without crick strand!

  The default alphabet is set to Biopython IUPACAmbiguousDNA

  The shape of the fragment is set by either:

  linear = False, True

  or

  circular = True, False

  Note that both ends of the DNA fragment has to be blunt to set circular = True (or linear = False).

      >>> pydna.Dseq("aaa","ttt")
      Dseq(-3)
      aaa
      ttt
      >>> pydna.Dseq("aaa","ttt",ovhg=0)
      Dseq(-3)
      aaa
      ttt
      >>> pydna.Dseq("aaa", "ttt", linear = False ,ovhg=0)
      Dseq(o3)
      aaa
      ttt
      >>> pydna.Dseq("aaa", "ttt", circular = True , ovhg=0)
      Dseq(o3)
      aaa
      ttt

  Coercing to string

      >>> a=pydna.Dseq("tttcccc","aaacccc")
      >>> a
      Dseq(-11)
          tttcccc
      ccccaaa

      >>> str(a)
      'ggggtttcccc'

  The double stranded part is accessible through the dsdata property:

      >>> a.dsdata
      'ttt'

  Dseqrecord and Dseq share the same concept of length:

  > <– length –> GATCCTTT

  > > AAAGCCTAG

  Methods

  `T4`(nucleotides=None)

    Fill in of five prime protruding ends and chewing back of three prime protruding ends by a DNA polymerase providing both 5’-3’ DNA polymerase activity and 3’-5’ nuclease acitivty (such as T4 DNA polymerase). This in presence of any combination of A, G, C or T. Default are all four nucleotides together.

    Examples

        >>> import pydna
        >>> a=pydna.Dseq("gatcgatc")
        >>> a
        Dseq(-8)
        gatcgatc
        ctagctag
        >>> a.T4()
        Dseq(-8)
        gatcgatc
        ctagctag
        >>> a.T4("t")
        Dseq(-8)
        gatcgat
         tagctag
        >>> a.T4("a")
        Dseq(-8)
        gatcga
          agctag
        >>> a.T4("g")
        Dseq(-8)
        gatcg
           gctag
        >>>

  `circular`

    The circular property

  `cut`(*enzymes)

    Returns a list of linear Dseq fragments produced in the digestion. If there is not cut, the whole sequence is returned.

  `fig`()

    Returns a representation of the sequence, truncated if longer than 40 bp:

    Examples

        >>> import pydna
        >>> a=pydna.Dseq("atcgcttactagcgtactgatcatctgactgactagcgtga")
        >>> a
        Dseq(-41)
        atcgcttactagcgtactga...catctgactgactagcgtga
        tagcgaatgatcgcatgact...gtagactgactgatcgcact
        >>>

  `fill_in`(nucleotides=None)

    Fill in of five prime protruding end with a DNA polymerase that has only DNA polymerase activity (such as exo-klenow) and any combination of A, G, C or T. Default are all four nucleotides together.

    Examples

        >>> import pydna
        >>> a=pydna.Dseq("aaa", "ttt")
        >>> a
        Dseq(-3)
        aaa
        ttt
        >>> a.fill_in()
        Dseq(-3)
        aaa
        ttt
        >>> b=pydna.Dseq("caaa", "cttt")
        >>> b
        Dseq(-5)
        caaa
         tttc
        >>> b.fill_in()
        Dseq(-5)
        caaag
        gtttc
        >>> b.fill_in("g")
        Dseq(-5)
        caaag
        gtttc
        >>> b.fill_in("tac")
        Dseq(-5)
        caaa
         tttc
        >>> b=pydna.Dseq("aaac", "tttg")
        >>> c=pydna.Dseq("aaac", "tttg")
        >>> c
        Dseq(-5)
         aaac
        gttt
        >>> c.fill_in()
        Dseq(-5)
         aaac
        gttt
        >>>

  `five_prime_end`()

    Returns a tuple describing the structure of the 5’ end of the DNA fragment

        >>> import pydna
        >>> a=pydna.Dseq("aaa", "ttt")
        >>> a
        Dseq(-3)
        aaa
        ttt
        >>> a.five_prime_end()
        ('blunt', '')
        >>> a=pydna.Dseq("aaa", "ttt", ovhg=1)
        >>> a
        Dseq(-4)
         aaa
        ttt
        >>> a.five_prime_end()
        ("3'", 't')
        >>> a=pydna.Dseq("aaa", "ttt", ovhg=-1)
        >>> a
        Dseq(-4)
        aaa
         ttt
        >>> a.five_prime_end()
        ("5'", 'a')
        >>>

  `linear`

    The linear property

  `looped`()

    Returns a circularized Dseq object. This can only be done if the two ends are compatible, otherwise a TypeError is raised.

    Examples

        >>> import pydna
        >>> a=pydna.Dseq("catcgatc")
        >>> a
        Dseq(-8)
        catcgatc
        gtagctag
        >>> a.looped()
        Dseq(o8)
        catcgatc
        gtagctag
        >>> a.T4("t")
        Dseq(-8)
        catcgat
         tagctag
        >>> a.T4("t").looped()
        Dseq(o7)
        catcgat
        gtagcta
        >>> a.T4("a")
        Dseq(-8)
        catcga
          agctag
        >>> a.T4("a").looped()
        Traceback (most recent call last):
          File "<stdin>", line 1, in <module>
          File "/usr/local/lib/python2.7/dist-packages/pydna/dsdna.py", line 357, in looped
            if type5 == type3 and str(sticky5) == str(rc(sticky3)):
        TypeError: DNA cannot be circularized.
        5' and 3' sticky ends not compatible!
        >>>

  `mung`()

    Simulates treatment a nuclease with 5’-3’ and 3’-5’ single strand specific exonuclease activity (such as mung bean nuclease):

            ggatcc    ->     gatcc
             ctaggg          ctagg

             ggatcc   ->      ggatc
            tcctag            cctag

        >>> import pydna
        >>> b=pydna.Dseq("caaa", "cttt")
        >>> b
        Dseq(-5)
        caaa
         tttc
        >>> b.mung()
        Dseq(-3)
        aaa
        ttt
        >>> c=pydna.Dseq("aaac", "tttg")
        >>> c
        Dseq(-5)
         aaac
        gttt
        >>> c.mung()
        Dseq(-3)
        aaa
        ttt

  `ovhg`

    The ovhg property

  `rc`()

    Alias of the reverse_complement method

  `reverse_complement`()

    Returns a Dseq object where watson and crick have switched places.

  `t4`(*args, **kwargs)

    Alias for the T4 method

  `three_prime_end`()

    Returns a tuple describing the structure of the 5’ end of the DNA fragment

        >>> import pydna
        >>> a=pydna.Dseq("aaa", "ttt")
        >>> a
        Dseq(-3)
        aaa
        ttt
        >>> a.three_prime_end()
        ('blunt', '')
        >>> a=pydna.Dseq("aaa", "ttt", ovhg=1)
        >>> a
        Dseq(-4)
         aaa
        ttt
        >>> a.three_prime_end()
        ("3'", 'a')
        >>> a=pydna.Dseq("aaa", "ttt", ovhg=-1)
        >>> a
        Dseq(-4)
        aaa
         ttt
        >>> a.three_prime_end()
        ("5'", 't')
        >>>

  `tolinear`()

    Returns a blunt, linear copy of a circular Dseq object. This can only be done if the Dseq object is circular, otherwise a TypeError is raised.

    Examples

        >>> import pydna
        >>> a=pydna.Dseq("catcgatc", circular=True)
        >>> a
        Dseq(o8)
        catcgatc
        gtagctag
        >>> a.tolinear()
        Dseq(-8)
        catcgatc
        gtagctag
        >>>

class `pydna.dsdna.``Dseqrecord`(record, circular=None, linear=None, *args, **kwargs)

  Bases: `Bio.SeqRecord.SeqRecord`

  Dseqrecord is a double stranded version of the Biopython SeqRecord class. The Dseqrecord object holds a Dseq object describing the sequence. Additionally, Dseqrecord hold meta information about the sequence in the from of a list of SeqFeatures, in the same way as the SeqRecord does. The Dseqrecord can be initialized with a string, Seq, Dseq, SeqRecord or another Dseqrecord. The sequence information will be stored in a Dseq object in all cases. Dseqrecord objects can be read or parsed from sequences in Fasta, Embl or Genbank format.

  There is a short representation associated with the Dseqrecord. `Dseqrecord(-3)` represents a linear sequence of length 2 while `Dseqrecord(o7)` represents a circular sequence of length 7.

  Dseqrecord and Dseq share the same concept of length:

      <-- length -->
      GATCCTTT
           AAAGCCTAG

  Parameters : | record : string, Seq, SeqRecord, Dseq or other Dseqrecord object This data will be used to form the seq property circular : bool, optional True or False reflecting the shape of the DNA molecule linear : bool, optional True or False reflecting the shape of the DNA molecule

  Examples

      >>> from pydna import Dseqrecord
      >>> a=Dseqrecord("aaa")
      >>> a
      Dseqrecord(-3)
      >>> a.seq
      Dseq(-3)
      aaa
      ttt
      >>> from Bio.Seq import Seq
      >>> b=Dseqrecord(Seq("aaa"))
      >>> b
      Dseqrecord(-3)
      >>> b.seq
      Dseq(-3)
      aaa
      ttt
      >>> from Bio.SeqRecord import SeqRecord
      >>> c=Dseqrecord(SeqRecord(Seq("aaa")))
      >>> c
      Dseqrecord(-3)
      >>> c.seq
      Dseq(-3)
      aaa
      ttt
      >>> a.seq.alphabet
      IUPACAmbiguousDNA()
      >>> b.seq.alphabet
      IUPACAmbiguousDNA()
      >>> c.seq.alphabet
      IUPACAmbiguousDNA()
      >>>

  Methods

  `circular`

    Not really a method, but the circular property

  `cut`(*enzymes)

    Digest the Dseqrecord object with one or more restriction enzymes. returns a list of linear Dseqrecords.

    Parameters : | enzymes : iterable object iterable containing Biopython restriction enzyme objects
    Returns : | fragments : list list of Dseqrecord objects formed by the digestion

    Examples

        >>> import pydna
        >>> a=pydna.Dseqrecord("ggatcc")
        >>> from Bio.Restriction import BamHI
        >>> a.cut(BamHI)
        [Dseqrecord(-5), Dseqrecord(-5)]
        >>> frag1, frag2 = a.cut(BamHI)
        >>> frag1.seq
        Dseq(-5)
        g
        cctag
        >>> frag2.seq
        Dseq(-5)
        gatcc
            g

  `format`(f='gb')

    Returns the sequence as a string using a format supported by Biopython SeqIO. Default is “gb” which is short for Genbank.

    Examples

        >>> import pydna
        >>> a=pydna.Dseqrecord("aaa")
        >>> a.annotations['date'] = '02-FEB-2013'
        >>> a
        Dseqrecord(-3)
        >>> print a.format()
        LOCUS       .                          3 bp    DNA     linear   UNK 02-FEB-2013
        DEFINITION  .
        ACCESSION   <unknown id>
        VERSION     <unknown id>
        KEYWORDS    .
        SOURCE      .
          ORGANISM  .
                    .
        FEATURES             Location/Qualifiers
        ORIGIN
                1 aaa
        //

  `linear`

    Not really a method, but the linear property

  `looped`()

    Returns a circular version of the Dseqrecord object. The underlying Dseq object has to have compatible ends.

    Examples

        >>> import pydna
        >>> a=pydna.Dseqrecord("aaa")
        >>> a
        Dseqrecord(-3)
        >>> b=a.looped()
        >>> b
        Dseqrecord(o3)
        >>>

  `rc`()

    alias of the reverse_complement method

  `reverse_complement`()

    Returns a new Dseqrecord object which is the reverse complement.

    Examples

        >>> import pydna
        >>> a=pydna.Dseqrecord("ggaatt")
        >>> a
        Dseqrecord(-6)
        >>> a.seq
        Dseq(-6)
        ggaatt
        ccttaa
        >>> a.reverse_complement().seq
        Dseq(-6)
        aattcc
        ttaagg
        >>>

  `seguid`()

    Returns the SEGUID for the sequence

    Examples

        >>> import pydna
        >>> a=pydna.Dseqrecord("aaa")
        >>> a.seguid()
        'YG7G6b2Kj/KtFOX63j8mRHHoIlE'

  `shifted`(shift)

    Returns a circular Dseqrecord with a new origin <shift>. This only works on circular Dseqrecords. If we consider the following circular sequence:

    `GAAAT <-- watson strand`
    `CTTTA <-- crick strand`

    The T and the G on the watson strand are linked together as well as the A and the C of the of the crick strand.

    if `shift` is 1, this indicates a new origin at position 1:

    new origin
    `G|AAAT`
    `C|TTTA`

    new sequence:

    `AAATG`
    `TTTAC`

    Shift is always positive and 0<shift<length, so in the example below, permissible values of shift are 1,2 and 3

        >>> import pydna
        >>> a=pydna.Dseqrecord("aaat",circular=True)
        >>> a
        Dseqrecord(o4)
        >>> a.seq
        Dseq(o4)
        aaat
        ttta
        >>> b=a.shifted(1)
        >>> b
        Dseqrecord(o4)
        >>> b.seq
        Dseq(o4)
        aata
        ttat

  `stamp`()

    Adds a seguid stamp to the description property. This will show in the genbank format. The following string:

    `SEGUID <seguid>`

    will be appended to the description property of the Dseqrecord object (string).

    Examples

        >>> import pydna
        >>> a=pydna.Dseqrecord("aaa")
        >>> a.stamp()
        >>> a.description
        '<unknown description> SEGUID YG7G6b2Kj/KtFOX63j8mRHHoIlE'
        >>> a.verify_stamp()
        True

  `synced`(ref, limit=25)

    This function returns a new circular sequence (Dseqrecord object), which has ben rotated in such a way that there is maximum overlap between the sequence and ref, which may be a string, Biopython Seq, SeqRecord object or another Dseqrecord object.

    The reason for using this could be to rotate a recombinant plasmid so that it starts at the same position after cloning. See the example below:

    Examples

        >>> import pydna
        >>> a=pydna.Dseqrecord("gaat",circular=True)
        >>> a.seq
        Dseq(o4)
        gaat
        ctta
        >>> d = a[2:] + a[:2]
        >>> d.seq
        Dseq(-4)
        atga
        tact
        >>> insert=pydna.Dseqrecord("CCC")
        >>> recombinant = (d+insert).looped()
        >>> recombinant.seq
        Dseq(o7)
        atgaCCC
        tactGGG
        >>> recombinant.synced(a).seq
        Dseq(o7)
        gaCCCat
        ctGGGta

  `tolinear`()

    Returns a linear, blunt of a circular Dseqrecord object. The underlying Dseq object has to be circular.

    Examples

        >>> import pydna
        >>> a=pydna.Dseqrecord("aaa", circular = True)
        >>> a
        Dseqrecord(o3)
        >>> b=a.tolinear()
        >>> b
        Dseqrecord(-3)
        >>>

  `verify_stamp`()

    Verifies the SEGUID stamp in the description property is valid. True if stamp match the sequid calculated from the sequence. Exception raised if no stamp can be found.

        >>> import pydna
        >>> a=pydna.Dseqrecord("aaa")
        >>> a.annotations['date'] = '02-FEB-2013'
        >>> a.seguid()
        'YG7G6b2Kj/KtFOX63j8mRHHoIlE'
        >>> print a.format("gb")
        LOCUS       .                          3 bp    DNA     linear   UNK 02-FEB-2013
        DEFINITION  .
        ACCESSION   <unknown id>
        VERSION     <unknown id>
        KEYWORDS    .
        SOURCE      .
          ORGANISM  .
                    .
        FEATURES             Location/Qualifiers
        ORIGIN
                1 aaa
        //
        >>> a.stamp()
        >>> a
        Dseqrecord(-3)
        >>> print a.format("gb")
        LOCUS       .                          3 bp    DNA     linear   UNK 02-FEB-2013
        DEFINITION  <unknown description> SEGUID YG7G6b2Kj/KtFOX63j8mRHHoIlE
        ACCESSION   <unknown id>
        VERSION     <unknown id>
        KEYWORDS    .
        SOURCE      .
          ORGANISM  .
                    .
        FEATURES             Location/Qualifiers
        ORIGIN
                1 aaa
        //
        >>> a.verify_stamp()
        True
        >>>

  `write`(filename=None, f='gb')

    Writes the Dseqrecord to a file using the format f, which must be a format supported by Biopython SeqIO. Default is “gb” which is short for Genbank.

    Filename is the path to the file where the sequece is to be written. The filename is optional, if it is not given, the description property (string) is used together with the format:

    If obj is the Dseqrecord object, the default file name will be:

    `<obj.description>.<f>`

    If the filename already exists and the sequence it contains is different, a new file name will be used:

    `<obj.description>_NEW.<f>`

`pydna.dsdna.``parse`(data, filter=False, ds=True)

  This function returns all DNA sequences found in data. If no sequences are found, an empty list is returned. This is a greedy function, use carefully.

  Parameters : | data : string or iterable see below filter : bool if filter == True, sequences will be silently filtered for allowed characters (see docs for Dseqrecord) ds : bool Double stranded or single stranded DNA, Return “Dseqrecord” or “SeqRecord” objects.
  Returns : | list : contains Dseqrecord or SeqRecord objects

  Notes

  The data parameter is a string containing:

  1. an absolute path to a local file. The file will be read in text mode and parsed for EMBL, FASTA and Genbank sequences.
  2. an absolute path to a local directory. all files in the directory will be read and parsed as in 1.
  3. a string containing one or more sequences in EMBL, GENBANK, or FASTA format. Mixed formats are allowed.
  4. data can be a list or other iterable of 1 - 3

`pydna.dsdna.``rc`(sequence)

  returns the reverse complement of sequence (string) accepts mixed DNA/RNA

`pydna.dsdna.``read`(data, filter=False, ds=True)

  This function is similar the parse funtion but returns only the first sequence found.

  Parameters : | data : string see below filter : bool if filter == True, sequences will be silently filtered for allowed characters (see docs for Dseqrecord) ds : bool Double stranded or single stranded DNA, Return “Dseqrecord” or “SeqRecord” objects.
  Returns : | Dseqrecord : contains the first Dseqrecord or SeqRecord object parsed.

  See also

  `parse`

  Notes

  The data parameter is similar to the data parameter for parse.

## `amplify` Module

This module provides functions for PCR.

Primers with 5’ tails as well as inverse PCR on circular templates are handled correctly.

class `pydna.amplify.``Amplicon`(forward_primer, reverse_primer, template, saltc=50, fc=1000, rc=1000)

  Holds information about a PCR reaction involving two primers and one template. This class is used by the Anneal class and is not meant to be instantiated directly.

  Parameters : | forward_primer : SeqRecord(Biopython) SeqRecord object holding the forward (sense) primer reverse_primer : SeqRecord(Biopython) SeqRecord object holding the reverse (antisense) primer template : Dseqrecord Dseqrecord object holding the template (circular or linear) saltc : float, optional saltc = monovalent cations (mM) (Na,K..) default value is 50mM This is used for Tm calculations. fc : float, optional primer concentration (nM) default set to 1000nM = 1µM This is used for Tm calculations. rc : float, optional primer concentration (nM) default set to 1000nM = 1µM This is used for Tm calculations.

  Methods

  `detailed_figure`()

        5gctactacacacgtactgactg3
         |||||||||||||||||||||| tm 52.6 (dbd) 58.3
        5gctactacacacgtactgactg...caagatagagtcagtaaccaca3
        3cgatgatgtgtgcatgactgac...gttctatctcagtcattggtgt5
                                  |||||||||||||||||||||| tm 49.1 (dbd) 57.7
                                 3gttctatctcagtcattggtgt5

    Returns : | figure:string : A string containing a text representation of the primers annealing on the template.

    Notes

    tm is the melting temperature (Tm) calculated according to SantaLucia 1998 [1] for each primer.

    (dbd) is Tm calculation for enzymes with dsDNA binding domains like Pfu-Sso7d [2]. See [3] for more information.

    References

    [R1] | SantaLucia, “A Unified View of Polymer, Dumbbell, and Oligonucleotide DNA Nearest-neighbor Thermodynamics,” Proceedings of the National Academy of Sciences 95, no. 4 (1998): 1460.

    [R2] | Nørholm, “A Mutant Pfu DNA Polymerase Designed for Advanced Uracil-excision DNA Engineering,” BMC Biotechnology 10, no. 1 (2010): 21, doi:10.1186/1472-6750-10-21.

    [R3] | http://www.thermoscientificbio.com/webtools/tmc/

  `flankdn`(flankdnlength=50)

    Returns a Dseqrecord object containing flankdnlength bases downstream of the reverse primer footprint. Truncated if the template is not long enough.

                                       <---- flankdn ------>

                        3actactgactatctTAATAA5
                         ||||||||||||||
        acgcattcagctactgtactactgactatctatcgtacatgtactatcgtat

  `flankup`(flankuplength=50)

    Returns a Dseqrecord object containing flankuplength bases upstream of the forward primer footprint, Truncated if the template is not long enough.

        <--- flankup --->

                  5TAATAAactactgactatct3
                         ||||||||||||||
        acgcattcagctactgtactactgactatctatcg

  `pcr_product`()

    Returns the PCR product as a Dseqrecord object. Primers are marked as SeqFeatures(Biopython) associated with the sequence.

  `pcr_program`()

    Returns a string containing a text representation of two proposed PCR programs. The first program is adapted for Taq poymerase, while the second is adapted for Pfu-Sso7d.

        Taq (rate 30 nt/s)
        Three-step|         30 cycles     |      |SantaLucia 1998
        94.0°C    |94.0°C                 |      |SaltC 50mM
        __________|_____          72.0°C  |72.0°C|
        04min00s  |30s  \         ________|______|
                  |      \ 46.0°C/ 0min 1s|10min |
                  |       \_____/         |      |
                  |         30s           |      |4-8°C

        Pfu-Sso7d (rate 15s/kb)
        Three-step|          30 cycles   |      |Breslauer1986,SantaLucia1998
        98.0°C    |98.0°C                |      |SaltC 50mM
        __________|_____          72.0°C |72.0°C|Primer1C   1µM
        00min30s  |10s  \ 61.0°C ________|______|Primer2C   1µM
                  |      \______/ 0min 0s|10min |
                  |        10s           |      |4-8°C

class `pydna.amplify.``Anneal`(primers, template, homology_limit=13, max_product_size=15000)

  Parameters : | primers : iterable containing SeqRecord objects Primer sequences 5’-3’. template : Dseqrecord object The template sequence 5’-3’. homology_limit : int, optional limit length of the annealing part of the primers. max_product_size: int, optional : PCR products longer than max_product_size are not reported

  Examples

      >>> import pydna
      >>> template = pydna.Dseqrecord("tacactcaccgtctatcattatctactatcgactgtatcatctgatagcac")
      >>> from Bio.SeqRecord import SeqRecord
      >>> p1=pydna.read(">p1\ntacactcaccgtctatcattatc", ds = False)
      >>> p2 = pydna.read(">p1\ncgactgtatcatctgatagcac", ds = False).reverse_complement()
      >>> pydna.Anneal((p1,p2), template)
      Anneal(amplicons = 1)
      >>> pydna.Anneal((p1,p2), template).amplicons
      [Amplicon(51)]
      >>> amplicon_list = pydna.Anneal((p1,p2), template).amplicons
      >>> amplicon = amplicon_list.pop()
      >>> amplicon
      Amplicon(51)
      >>> print amplicon.detailed_figure()
      5tacactcaccgtctatcattatc...cgactgtatcatctgatagcac3
                                 |||||||||||||||||||||| tm 50.6 (dbd) 60.5
                                3gctgacatagtagactatcgtg5
      5tacactcaccgtctatcattatc3
       ||||||||||||||||||||||| tm 49.4 (dbd) 58.8
      3atgtgagtggcagatagtaatag...gctgacatagtagactatcgtg5
      >>> prod = amplicon.pcr_product()
      >>> prod.annotations['date'] = '02-FEB-2013'
      >>> print prod
      Dseqrecord
      circular: False
      size: 51
      ID: 51bp U96+TO06Y6pFs74SQx8M1IVTBiY
      Name: 51bp_PCR_prod
      Description: Primers p1 <unknown name>
      Number of features: 2
      /date=02-FEB-2013
      Dseq(-51)
      tacactcaccgtctatcatt...actgtatcatctgatagcac
      atgtgagtggcagatagtaa...tgacatagtagactatcgtg
      >>>

  Attributes

  Methods

  `featured_template`()

    returns the template Dseqrecord object with the footprint of all annealing primers marked as SeqFeatures.

  `products`()

    returns all pcr product as a list of Dseqrecords

  `report`()

    This method is an alias of str(Annealobj). Returns a short string representation.

`pydna.amplify.``basictm`(primer)

  Returns the melting temperature (Tm) of the primer using the basic formula.

  Tm = (wA+xT)*2 + (yG+zC)*4 assumed 50mM monovalent cations
  w = number of A in primer
  x = number of T in primer
  y = number of G in primer
  z = number of C in primer

  Parameters : | primer : string Primer sequence 5’-3’
  Returns : | tm : int

  Examples

      >>> from pydna.amplify import basictm
      >>> basictm("ggatcc")
      20
      >>>

`pydna.amplify.``pcr`(*args, **kwargs)

  pcr is a convenience function for Anneal to simplify its usage, especially from the command line. If more than one PCR product is formed, an exception is raised.

  args is any iterable of sequences or an iterable of iterables of sequences. args will be greedily flattened.

  Parameters : | args : iterable containing sequence objects Several arguments are also accepted. limit : int = 13, optional limit length of the annealing part of the primers.
  Returns : | product : Dseqrecord a Dseqrecord object representing the PCR product. The direction of the PCR product will be the same as for the template sequence.

  Notes

  sequences in args could be of type:

  string Seq SeqRecord Dseqrecord

  The last sequence will be interpreted as the template all preceeding sequences as primers.

  This is a powerful function, use with care!

  Examples

      >>> import pydna
      >>> template = pydna.Dseqrecord("tacactcaccgtctatcattatctactatcgactgtatcatctgatagcac")
      >>> from Bio.SeqRecord import SeqRecord
      >>> p1 = pydna.read(">p1\ntacactcaccgtctatcattatc", ds = False)
      >>> p2 = pydna.read(">p1\ncgactgtatcatctgatagcac", ds = False).reverse_complement()
      >>> pydna.pcr(p1, p2, template)
      Dseqrecord(-51)
      >>> pydna.pcr([p1, p2], template)
      Dseqrecord(-51)
      >>> pydna.pcr((p1,p2,), template)
      Dseqrecord(-51)
      >>>

`pydna.amplify.``tmbreslauer86`(primer, dnac=500.0, saltc=50.0, thermodynamics=False)

  Returns the melting temperature (Tm) of the primer using the nearest neighbour algorithm. Formula and thermodynamic data is taken from Breslauer 1986. These data are no longer widely used.

  Breslauer 1986, table 2 [1]

  pair | dH | dS | dG
  --- | --- | --- | ---
  AA/TT | 9.1 | 24.0 | 1.9
  AT/TA | 8.6 | 23.9 | 1.5
  TA/AT | 6.0 | 16.9 | 0.9
  CA/GT | 5.8 | 12.9 | 1.9
  GT/CA | 6.5 | 17.3 | 1.3
  CT/GA | 7.8 | 20.8 | 1.6
  GA/CT | 5.6 | 13.5 | 1.6
  CG/GC | 11.9 | 27.8 | 3.6
  GC/CG | 11.1 | 26.7 | 3.1
  GG/CC | 11.0 | 26.6 | 3.1

  Parameters : | primer : string Primer sequence 5’-3’ in UPPERCASE
  Returns : | tm : float

  References

  [R4] | K.J. Breslauer et al., “Predicting DNA Duplex Stability from the Base Sequence,” Proceedings of the National Academy of Sciences 83, no. 11 (1986): 3746.

  Examples

      >>> from pydna.amplify import tmbreslauer86
      >>> tmbreslauer86("ACGTCATCGACACTATCATCGAC")
      64.28863985851899

`pydna.amplify.``tmbresluc`(primer, primerc=500.0, saltc=50.0, thermodynamics=False)

  Returns the tm for a primer using a formula adapted to polymerases with a DNA binding domain.

  Parameters : | primer : string primer sequence 5’-3’ primerc : float concentration (nM) saltc : float, optional Monovalent cation concentration (mM) thermodynamics : bool, optional prints details of the thermodynamic data to stdout. For debugging only.
  Returns : | tm : float the tm of the primer tm,dH,dS : tuple tm and dH and dS used for the calculation

`pydna.amplify.``tmstaluc98`(primer, dnac=50, saltc=50)

  Returns the melting temperature (Tm) of the primer using the nearest neighbour algorithm. Formula and thermodynamic data is taken from SantaLucia 1998. This implementation gives the same answer as the one provided by Biopython (See Examples).

  Thermodynamic data used:

  pair | dH | dS
  --- | --- | ---
  AA/TT | 7.9 | 22.2
  AT/TA | 7.2 | 20.4
  TA/AT | 7.2 | 21.3
  CA/GT | 8.5 | 22.7
  GT/CA | 8.4 | 22.4
  CT/GA | 7.8 | 21.0
  GA/CT | 8.2 | 22.2
  CG/GC | 10.6 | 27.2
  GC/CG | 9.8 | 24.4
  GG/CC | 8.0 | 19.9

  Parameters : | primer : string Primer sequence 5’-3’ in UPPERCASE
  Returns : | tm : float tm of the primer

  References

  [R5] | SantaLucia, “A Unified View of Polymer, Dumbbell, and Oligonucleotide DNA Nearest-neighbor Thermodynamics,” Proceedings of the National Academy of Sciences 95, no. 4 (1998): 1460.

  Examples

      >>> from pydna.amplify import tmstaluc98
      >>> from Bio.SeqUtils.MeltingTemp import Tm_staluc
      >>> tmstaluc98("ACGTCATCGACACTATCATCGAC")
      54.55597724052518
      >>> Tm_staluc("ACGTCATCGACACTATCATCGAC")
      54.555977240525124
      >>>

## `assembly` Module

Provides functions for assembly of sequences by homologous recombination. Given a list of sequences (Dseqrecords), all sequences will be analyzed for overlapping regions of DNA (common substrings).

The assembly algorithm is based on forming a network where each overlapping sequence forms a node and intervening sequences form edges.

Then all possible linear or circular assemblies will be returned in the order of their length.

`pydna.assembly.``circular_assembly`(form_rec_list, limit=25)

  Accepts a list of Dseqrecords and tries to assemble them into a circular assembly by homologous recombination based on shared regions of homology with a minimum length given by limit.

  Parameters : | form_rec_list : list a list of Dseqrecord objects. limit : int, optional limit is set to 25 by default.
  Returns : | frecs, cp : tuple frecs are the same Dseqrecords as given as arguments, but with the regions of homology added to the features. cp is a list of Dseqrecords representing the circular products sorted by length (long -> short).

`pydna.assembly.``linear_assembly`(form_rec_list, limit=25)

  Accepts a list of Dseqrecords and tries to assemble them into a linear assembly by homologous recombination based on shared regions of homology with a minimum length given by limit.

  Parameters : | form_rec_list : list a list of Dseqrecord objects. limit : int, optional limit is set to 25 by default.
  Returns : | frecs, lp : tuple frecs are the same Dseqrecords as given as arguments, but with the regions of homology added to the features. lp is a list of Dseqrecords representing the linear products sorted by length (long -> short).

## `download` Module

Provides a class for downloading sequences from genbank.

class `pydna.download.``Genbank`(users_email, proxy=None, tool='pydna')

  Class to facilitate download from genbank.

  Parameters : | users_email : string Has to be a valid email address. You should always tell Genbanks who you are, so that they can contact you. proxy : string, optional String containing a proxy url: “proxy = “http://umiho.proxy.com:3128“ tool : string, optional Default is “pydna”. This is to tell Genbank which tool you are using.

  Methods

  `nucleotide`(item)

    Download a genbank record using a Genbank object.

    item is a string containing one genbank acession number [1] for a nucleotide file:

    A12345 = 1 letter + 5 numerals
    AB123456 = 2 letters + 6 numerals

    References

    [R6] | http://www.dsimb.inserm.fr/~fuchs/M2BI/AnalSeq/Annexes/Sequences/Accession_Numbers.htm

  `test`()

    Test downloading the pUC19 plasmid sequence from genbank returns True if successful. Can be used to test the proxy and other network settings.

## `editor` Module

This module provides a class for opening a sequence using an editor that accepts a path as a command line argument.

ApE - A plasmid Editor [R7] is such an editor.

### References

[R7] | http://biologylabs.utah.edu/jorgensen/wayned/ape/

### Examples

    >>> import pydna
    >>> ape = pydna.Editor("tclsh /home/bjorn/ApE/apeextractor/ApE.vfs/lib/app-AppMain/AppMain.tcl")
    >>> ape.open("aaa")
    >>>

class `pydna.editor.``Editor`(shell_command_for_editor, tmpdir=None)

  Methods

  `open`(*args, **kwargs)

    Open a sequence for editing in an editor.

    Parameters : | args : sequence or iterable of sequences sequences to to be opened by the editor.

## `utils` Module

This module provides miscellaneous functions.

`pydna.utils.``copy_features`(source_sr, target_sr, limit=10)

  This function tries to copy all features in source_seq and copy them to target_seq. Source_sr and target_sr are objects with a features property, such as Dseqrecord or Biopython SeqRecord.

  Parameters : | source_seq : SeqRecord or Dseqrecord The sequence to copy features from target_seq : SeqRecord or Dseqrecord The sequence to copy features to
  Returns : | bool : True This function acts on target_seq in place. No data is returned.

`pydna.utils.``eq`(*args, **kwargs)

  Compares two or more DNA sequences for equality i.e. they represent the same DNA molecule. Comparisons are case insensitive.

  Parameters : | args : iterable iterable containing sequences args can be strings, Biopython Seq or SeqRecord, Dseqrecord or dsDNA objects. circular : bool, optional Consider all molecules circular or linear linear : bool, optional Consider all molecules circular or linear
  Returns : | eq : bool Returns True or False

  Notes

  Compares two or more DNA sequences for equality i.e. if they represent the same DNA molecule.

  Two linear sequences are considiered equal if either:

  - They have the same sequence (case insensitive)
  - One sequence is the reverse complement of the other (case insensitive)

  Two circular sequences are considiered equal if:

  1. They have the same length.

  AND

  2. One sequence or its reverse complement can be found in the concatenation of the other sequence with itself (they are circular permutations).

  The topology for the comparison can be set using one of the keywords linear or circular to True or False.

  If circular or linear is not set, it will be deduced from the topology of each sequence for sequences that have a linear or circular attribute (like Dseq and Dseqrecord).

  Examples

      >>> from pydna import eq, Dseqrecord
      >>> eq("aaa","AAA")
      True
      >>> eq("aaa","AAA","TTT")
      True
      >>> eq("aaa","AAA","TTT","tTt")
      True
      >>> eq("aaa","AAA","TTT","tTt", linear=True)
      True
      >>> eq("Taaa","aTaa", linear = True)
      False
      >>> eq("Taaa","aTaa", circular = True)
      True
      >>> a=Dseqrecord("Taaa")
      >>> b=Dseqrecord("aTaa")
      >>> eq(a,b)
      False
      >>> eq(a,b,circular=True)
      True
      >>> a=a.looped()
      >>> b=b.looped()
      >>> eq(a,b)
      True
      >>> eq(a,b,circular=False)
      False
      >>> eq(a,b,linear=True)
      False
      >>> eq(a,b,linear=False)
      True
      >>> eq("ggatcc","GGATCC")
      True
      >>> eq("ggatcca","GGATCCa")
      True
      >>> eq("ggatcca","tGGATCC")
      True

`pydna.utils.``shift_origin`(seq, shift)

  Shift the origin of seq which is assumed to be a circular sequence.

  Parameters : | seq : string, Biopython Seq, Biopython SeqRecord, Dseq or Dseqrecord sequence to be shifted.
  Returns : | new_seq : string, Biopython Seq, Biopython SeqRecord, Dseq or Dseqrecord sequence with a new origin.

  Examples

      >>> import pydna
      >>> pydna.shift_origin("taaa",1)
      'aaat'
      >>> pydna.shift_origin("taaa",0)
      'taaa'
      >>> pydna.shift_origin("taaa",2)
      'aata'
      >>> pydna.shift_origin("gatc",2)
      'tcga'

`pydna.utils.``sync`(seq, ref)

  Synchronize two circular sequences.

  Parameters : | seq : string, Biopython Seq, Biopython SeqRecord, Dseq or Dseqrecord sequence to be shifted. ref : string, Biopython Seq, Biopython SeqRecord, Dseq or Dseqrecord The reference sequence.
  Returns : | sequence : string, Biopython Seq, Biopython SeqRecord, Dseq or Dseqrecord sequence with a new origin. This function tries to rotate the circular sequence seq : so that it has a maximum overlap with ref. :

  Examples

      >>> import pydna
      >>> pydna.sync("taaatc","aaataa")
      'aaatct'
      >>> pydna.sync("taaatc","aaataa")
      'aaatct'
      >>> pydna.sync("taaat","aaataa")
      'aaatt'

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